POLY(ADP-RIBOSYL)ATION IN TRYPANOSOMATIDS

BALTANÁS, RODRIGO; ALONSO, GUILLERMO D.; TORRES, HÉCTOR N.; FLAWIÁ, MIRTHA M.; FERNÁNDEZ VILLAMIL, SILVIA H.

Pinamar, Argentina.

Congreso; X Congress of the Pan-American Association for Biochemistry and Molecular Biology (PABMB), XLI Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology (SAIB) and the XX Annual Meeting of the Argentine Society for Neurochemistry (SAN; 2005

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

The poly(ADP-ribosyl)ation is a posttranslational modification were an ADP-ribose branched polymer is attached to proteins. This polymer is synthesized by the enzyme poly(ADP-ribose)polymerase (PARP) from the substrate NAD+. Two members of the PARP family are activated upon DNA damage PARP-1 and PARP-2. Searching Trypanosoma cruzi genome databases we found an ORF encoding for a PARP-2 homologue, which was cloned and expressed in Codon Plus BL 21(DE3) pLys E cells by using pET-Tc-PARP recombinant vector. We performed Southern, Northern and Western studies which reported a single copy gene; an mRNA of about 1.8 kb and a 65 kDa protein. A nuclear and kinetoplastic localization in epimastigotes was ascertained by IFI. The Tc-PARP was purified and it´s activity was found to be dependent on nicked DNA concentration but didn´t exhibit metal ion requirement. Tc-PARP was inhibited by nicotinamide, 3 aminobenzamide, theophylline and thymidine, as well as by Mn2+, Ni+, Zn2+ and ATP. We demonstrated an attachment of poly(ADP-ribose) chains, synthesized by Tc-PARP, to it-self. The Tc-PARP activity decreased due to automodification, and it was recovered after removing ADP-ribose polymer from the protein by alkaline treatment, thus suggesting a negative regulation by automodification. We have also found the catabolic counterpart, poly(ADP-ribose)glycohydrolase (PARG) in T. cruzi genome databases, suggesting an analogy with the known poly(ADP-ribose) metabolism in higher eukaryotes.